Duhuo Jisheng Decoction regulates intracellular zinc homeostasis by enhancing autophagy via PTEN/Akt/mTOR pathway to improve knee cartilage degeneration

Background Articular cartilage and cartilage matrix degradation are key pathological changes occurring in the early stage of knee osteoarthritis (KOA). However, currently, there are limited strategies for early prevention and treatment of KOA. Duhuo Jisheng Decoction (DHJSD) is a formula quoted in Bei Ji Qian jin Yao Fang, which was compiled by Sun Simiao in the Tang Dynasty of China. As a complementary therapy, it is widely used to treat early-stage KOA in China; however, its mechanism has not been completely elucidated. Objective This study investigated the potential role of DHJSD in preventing cartilage degradation and the underlying mechanism. Methods A rat model of KOA model was established via the Hulth method. Subsequently, 25 rats were randomized into sham (saline), model control (saline), high-DHJSD (1.9g/mL of DHJSD), medium-DHJSD (1.2g/mL of DHJSD), and low-DHJSD groups (0.6g/mL of DHJSD). After 4 weeks of treatment, all rats were sacrificed and the severity of the cartilage degeneration was evaluated by a series of histological methods. The autophagosome was observed using transmission electron microscopy, and the related functional proteins were detected by the western blotting and real-time polymerase chain reaction. Next, the mechanism by which DHJSD improves knee cartilage degeneration was further clarified the in vitro by gene silencing technology combined with a series of functional experiments. The proteins levels of PTEN, Akt, p-Akt, mTOR, and p-mTOR, as well as the marker proteins of autophagy and apoptosis were determined. Zinc levels in chondrocytes were determined using inductively coupled plasma mass spectrometry. Results Histopathological staining revealed that DHJSD had a protective effect on the cartilage. DHJSD increased autophagosome synthesis and the expression of autophagy proteins LC3 and Beclin-1 in chondrocytes. Moreover, it reduced the phosphorylation levels of Akt and mTOR and the levels of zinc, MMP-13, Bax, and Bcl-2. Following PTEN silencing, this DHJSD-mediated reduction in Akt and mTOR phosphorylation and Bax, Bcl-2, and zinc levels were further decreased; in addition, DHJSD-mediated increase in LC3 and Beclin-1 levels was decreased. Conclusion DHJSD inhibits the Akt/mTOR signaling pathway by targeting PTEN to promote autophagy in chondrocytes, which may help reduce MMP-13 production by regulating zinc levels in chondrocytes.


Introduction
Knee osteoarthritis (KOA) is a common clinical disease in orthopedic surgery, with a prevalence of 25.51% in China [1].Particularly in China, with an increase in population aging and average life expectancy, the prevalence of KOA is estimated to rise [2].Reportedly, KOA is caused by several factors including trauma, immune abnormalities, degeneration, and metabolic abnormalities.The late stage of KOA is characterized by irreversible bone hyperplasia, joint surface degeneration, and joint space narrowing.Conservative treatment is often ineffective, and surgery is almost the only option for the late stage of KOA [3].At present, knee arthroplasty is the most effective surgical method to reduce knee pain and improve knee function.However, this surgery is associated with the risk of surgical complications, such as surgical failure, infection, and anesthesia risk; in addition the cost of surgical treatment is relatively high [4].Therefore, early treatment of KOA is of great significance in disease management.The primary goals of the treatment of early KOA include delaying the degeneration of articular cartilage and relieving pain.Although opioids and non-steroidal anti-inflammatory drugs have definite effects in relieving pain, these are associated with a risk of gastrointestinal tract and cardiovascular adverse events [5].Moreover, the long-term clinical efficacy of diacerein and glucosamine is controversial [6][7][8].
Duhuo Jisheng Decoction (DHJSD) is a formula quoted in Bei Ji Qian jin Yao Fang, a Chinese medicine compilation by Sun Simiao from the Tang Dynasty of China.As a complementary therapy, DHJSD is widely for the management of bone and joint diseases in China; however, its exact mechanism has not been completely elucidated.Existing studies have confirmed that DHJSD regulates inflammatory factors, reduces apoptosis and pyroptosis of nucleus pulposus cells, and inhibits extracellular matrix degradation [9][10][11][12], indicating the multiple mechanisms of action of DHJSD in osteoarthritis (OA).
The pathogenesis of OA involves the joint action of several factors, with articular cartilage degeneration being primarily mediated by excessive degradation of the extracellular matrix [13].Notably, the degradation of chondrocytes matrix mediated by zinc is an important link in cartilage degeneration [14,15].It has been reported that intracellular zinc homeostasis plays an important role in autophagy [16][17][18].Therefore, autophagy may be closely associated with the regulation of zinc homeostasis in chondrocytes and may be an upstream mechanism of zinc-mediated cartilage degeneration.Studies have shown that DHJSD can reduce the level of intracellular zinc and interfere with the degradation of the cell matrix to induce a chondrocyte protective effect [19].
The PI3K/Akt/mTOR pathway is an important intracellular autophagy signal regulation pathway.Activation of this pathway can regulate cell growth and metabolism, inhibit activity of autophagy-related proteins, and reduce autophagy, which have implications for cell growth, energy metabolism, and pathogenesis of several diseases [20,21].In particular, the PI3K/Akt/ mTOR signaling pathway plays a critical role in autophagy among chondrocyte [21].LC3 is a molecular marker of autophagy that is located in the cytoplasm.During autophagy, LC3-I binds covalently to phosphatidyleolamine to form LC3-II and localizes on the autophagosome membrane to participate in the formation of autophagosome [22].Inhibition of this pathway has been reported to increase the level of autophagy [23].In addition, phosphatase and tensin homolog deleted on chromosome ten (PTEN) is a typical tumor suppressor gene that inhibits the PI3K/Akt/mTOR growth signaling cascade [24].This pathway can be regulated via multiple factors [25], and an alteration in these factors will lead to the dysregulation of this pathway and other pathways, leading to overgrowth [26].The PTEN protein comprises four heterogeneous structures, PTEN-L, PTEN-M, PTEN-N and PTEN-O; of these, PTEN-L plays an important role in regulating the PI3K/Akt/mTOR signaling pathway as well as autophagy [27,28].In addition, PTEN is a typical tumor suppressor gene that inhibits the PI3K/Akt/ mTOR signaling cascade, thereby increasing autophagy and reducing apoptosis [29].Accordingly, the present study evaluated the effect of DHJSD on cartilage and evaluated whether this effect was mediated via the PTEN/Akt/mTOR, autophagy, and zinc homeostasis in chondrocytes.

Animal experiments
Establishment of OA rat model.The animal study was approved by the Experimental Animal Ethics Committee of Hospital of Chengdu University of Traditional Chinese Medicine (No.2023DL-005).A total of 25 adult, male Wistar rats (weight:198g-220 g) were obtained from Dossy (Chengdu, Sichuan, China).The rats were acclimatized for 1 week at 24±2˚C under a 12-h light/dark cycle and were provided free access to food and water.Subsequently, rats were randomized into the sham control group (where only the joint cavity was exposed; n = 5), model control group (n = 5), high-dose (HD) group (n = 5), medium-dose (MD) group (n = 5), low-dose (LD) group (n = 5).
The rat OA model was established using the Hulth method after inducing anesthesia using intraperitoneal pentobarbital sodium [30].As showed in Fig 1 , OA was induced by incising the medial side of the right posterior knee joint of anesthetized rats.The muscles and ligaments were then separated to expose the joint cavity.The medial collateral ligament, anterior and posterior cruciate ligament, and the medial meniscus were cut off.Subsequently, the incision was sutured, and each rat was intramuscularly administered penicillin (400 000 U/d) for 3 consecutive days and housed in the cage.Animal treatments.The HD, MD, and LD groups were administered with 1.5 mL/100 g�day of high, medium, and low-dose DHJSD, respectively, via gavage twice daily for 4 weeks from the first day after operation.The sham and model control groups received saline at the same dosage as DHJSD.After treatment, all rats were euthanized in accordance with the ethical guidelines for animal welfare, and blood and knee joint specimens were obtained for further assessments.

Histological analysis and macroscopic observation
Cartilage morphology was evaluated using hematoxylin-eosin, Masson, immunohistochemical, and TRAP staining via light microscopy.The autophagosomes in chondrocytes were observed using transmission electronic microscopy.

Preparation of DHJSD-serum
Twenty adult, male Wistar rats (weight: 198g-220 g) were obtained from Chengdu Dossy Experimental Animals Co. LTD (Sichuan, China).After 1 week of acclimatizing under the aforementioned conditions, the animals were randomized into two groups: group A (n = 10) received 1.5 mL/100 g�day of DHJSD twice daily for 1 week and group B (n = 10) received the same dosage of normal saline.One hour after the last administration, blood was collected from the abdominal aorta, and the serum was separated by centrifugation.The obtained serum was inactivated in a water bath at 56˚C for 30 minutes, packaged, and frozen at -80˚C until further use.Subsequently, all rats were euthanized in accordance with the ethical guidelines for animal welfare.

Quality control of DHJSD
DHJSD components were detected using an LC-MS/MS system, ultra-high performance liquid chromatograph (Nexera UHPLC LC-30A, SHIMADZU, Japan) with mass spectrometer (Tri-pleTOF5600+, AB SCIEX™).The liquid chromatography conditions were as follows: Chrom-Core 120 C18 Column (1.8μm 2.1mm*150mm), column temperature of 40˚C, mobile phase A of 0.1% formic acid and mobile phase B of 100% ACN, flow rate of 0.3mL/min, and analysis time of 21min.Detection was performed using electrospray ionization (ESI) in negative ion mode.The ESI source conditions were as follows: Ion Source Gas1 (Gas 1): 50, Ion Source Gas2 (Gas 2): 50, Curtain Gas (CUR): 25, temperature of 450˚C (negative ion), voltage of 4400V (negative ion), TOF MS scan range of 100-1200Da, product ion scan range of 50-1000Da, TOF MS scan accumulation time of 0.2s, product ion scan accumulation time of 0.01s.The second-level mass spectrometry was obtained using information-dependent acquisition (IDA) in high sensitivity mode, with a declustering potential (DP) of ±60V and a collision energy of 35±15eV.

PTEN siRNA transfection
The chondrocytes were transfected with a specific PTEN siRNA obtained from RiboBio (Guangzhou, China).For this, 15 μL of siRNA was diluted with 360 μL 1X riboFECT TM CP Buffer in a 1.5 mL centrifuge tube and gently mixed.To this, 36 μL of the riboFECT TM CP Reagent was added, gently blown, and mixed, followed by incubation at room temperature for 15 min to prepare the riboFECT TM CP transfection complex (siRNA concentration: 50 nmol).Chondrocytes were seeded in a six-well plate.After ensuring cell adhesion to the wall, the supernatant in the wells was removed by suction.Subsequently, the riboFECT TM CP transfection complex was added to an appropriate amount of the complete medium without double antibody, mixed, added to the well plate, and then incubated at 37˚C and 5% CO 2 for further use.

Cell treatment and CCK-8 assay
The CCK-8 assay was used to determine the optimal concentration of DHJSD-serum (Ds) obtained using four doses (0%, 5%, 10%, 15%, and 20%).The CCK-8 kit (B3350A, 21169949, Spec.5*100T, Biosharp, Anhui, China) was used to analyze the cell proliferation rate, in accordance with the manufacturer's instructions.The primary rat chondrocytes (Rat-iCell-s003, iCell) (5×104/mL, 100 μL/ well) in each group (4 wells in each group) were seeded in 96-well plates (edge wells were filled with sterile phosphate-buffered saline) and cultured for 48 h at 37˚C with 5% CO 2 .After 48 h of treatment, the supernatant was aspirated and discarded.Subsequently, the CCK-8 reagent was diluted with a serum-free medium at a ratio of 1:10, and 110 μL of the diluted CCK-8 solution/well was added to the plates.The culture plate was then gently shaken several times and incubated further for 2 h at 37˚C and 5% CO 2 .The absorbance values of each well were measured at 450 nm using a microplate reader.

Measurement of cell apoptosis
The cells in each group were collected and resuspended in 500 μL binding Buffer.The 5 μL of Annexin V-APC/PI (Kit: KGA1030, KeyGEN, Jiangsu, China) was added to blow gently, followed by the addition of 5 μL of propidium iodide.The reaction was conducted at room temperature in the dark for 15min, following which apoptosis was detected and analyzed.

Determination of intracellular zinc levels
The sample was placed in a Teflon vessel according to the grouping and concentrated nitric acid (5 mL) was added.Subsequently, the sample was digested using a microwave digestion apparatus (Shanghai, China), as per the standard protocol of the machine.Using the tuning fluid, instrument parameters were adjusted and optimized before each experiment to meet the requirements of sensitivity, resolution, and stability.The analysis was conducted in the CCT (He/O 2 ) mode.The main working parameters and acquisition conditions of the instrument are listed below [31,32].Tuning mode: STD/KED; dwell time: 0.1 s; peristaltic pump speed: 40 rpm; sample introduction time: 40 s; plasma power: 1550 W; sampling depth: 5.0 mm; nebulizer flow:0.98L/min; cool flow:14.0L/min; auxilliary flow:0.8L/min; spray chamber temperature: 2.7˚C; torch horizontal position: 0.16 mm; torch vertical position: -0.53 mm; helium flow: 4.55 mL/min; oxygen flow: 0.3125 mL/min; D1 lens: -350 V; and D2 lens: -350 V; repeat times: 3 times.The reference, multi-element calibration standard, and internal standard were purchased from the National Center of Analysis and Testing for Nonferrous Metals and Electronic Materials (Beijing, China).

Real-time polymerase chain reaction
Total cellular RNA was isolated using animal total RNA isolation kit (RE-03014, Spec.200, Foregene, China) according to the manufacture's instruction.Complementary DNA (cDNA) was reverse-transcribed with PrimeScript-RT reagent kit (RR047A, Spec.100,Takara, Japan).The mRNA levels of Beclin-1, Bax, Bcl-2 and PTEN were detected by RT-PCR with TB Green TM Premix Ex TaqTM II(Tli RNaseH Plus) (RR820A, Spec.200, Takara, Japan).The complete gene sequences showed in Table 2 were searched from the National Center for Biotechnology Information (NCBI) database, and the specific primers were designed and screened by Primer Premier software.All primers were designed and synthesized by Sangon Bioengineering Technology(Shanghai, China), and purified by ULTRAPAGE.Data were analyzed by 2 −ΔΔCT method.

Western blot analysis
The protein levels of PTEN, Akt, p-Akt, mTOR, p-mTOR, Bax, Bcl-2, Beclin-1 and LC3-II/I in chondrocytes were detected using western blotting.Total cellular proteins were extracted using RIPA lysates.The proteins were transferred to PVDF membrane by wet rotation method and blocked at room temperature for 1 h.The primary antibody was incubated overnight and the membrane was washed 3 times with TBST for 5 min each time.The secondary antibody was incubated at room temperature for 2 h, and the membrane was washed 3 times with TBST for 10 min each time.Then the ECL developer solution was uniformly added to the membrane for exposure development.The bands were scanned by exposure using Tianneng GIS chassis control software V2.0, and the results were expressed as the relative expression of the target protein.

Statistical analyses
SPSS 26 (IBM1 SPSS1 Statistics) was used to analyze the data in this study, and figures were plotted using GraphPad Prism v9.5.1 (GraphPad Software, Inc.).All data are presented as the means ± standard error of means, and all experiments were performed in triplicate.Differences among groups were analyzed by one-way analysis of variance, followed by Fisher's post hoc test.Differences between two groups were analyzed using the unpaired, two-tailed Student's t-test.Results with P<0.05 were considered statistically significant.

Qualitative analysis of DHJSD components
Liquid-chromatography tandem mass spectrometric analysis of DHJSD revealed 173 compounds.Compounds with a total score of 100 as well as their formula, ontology, reference and retention time are listed as in Table 3. Positive and negative ion chromatograms were provided in the Supplementary Material (S1 Fig).

Effects of DHJSD on MMP-13 and IL-1β in KOA rats
Studies have shown that DHJSD inhibits the production of inflammatory factors, including interleukins and matrix metalloproteinases (MMPs) [33,34].However, there is a lack of detailed research on the mechanism via which DHJSD in protects articular cartilage in KOA.
In the present study, the rat KOA model was established using the classic Hulth method.Unlike previous studies, the current study administered DHJSD to rats 24 h after surgery.Regarding the inflammatory factors (Fig 2 ), the levels of MMP-13 and IL-1β in the serum, as well as the expression area of MMP-13 in the cartilage matrix were increased in four weeks after KOA induction.However, pretreatment with DHJSD decreased these levels by varying degrees in KOA rats, and the differences were statistically significant.These results further confirm that DHJSD can reduce the expression of MMP-13 in the cartilage matrix and serum of rats with KOA.

DHJSD alleviated cartilage pathological characteristics in KOA rats
Next, we examined the effects of DHJSD on cartilage morphology in rats.Hematoxylin-eosin staining, Masson staining, immunohistochemical staining and TRAP staining were used to evaluate the pathological characteristics of cartilage.After 4 weeks of KOA induction, the model group exhibited severe loss of type II collagen, increased cartilage destruction, osteoclast formation, and collagen fiber formation, and higher Mankin score than the sham control group (Fig 3).However, pretreatment with HD DHJSD resulted in only mild changes.The results indicated that DHJSD had a certain inhibitory effect on cartilage destruction.

DHJSD increased autophagy and inhibited apoptosis in KOA rats
As

DHJSD modulated the PTEN/Akt/mTOR signaling pathway in KOA rats
It is well known that the Akt/mTOR signaling axis is closely related to autophagy and that PTEN has a direct regulatory effect on this signaling pathway.We hypothesized that DHJSD-

DHJSD increased autophagy and reduced apoptosis and zinc levels in vitro
The results of in vivo experiments confirmed that DHJSD improved cartilage degeneration in KOA rats by inducing autophagy in chondrocytes.Accordingly, we further attempted to clarify the effects of DHJSD on the chondrocytes using in vitro experiments.Based on in vivo data, HD DHJSD was used to prepare the Ds, as shown in Fig 6A.To first determine the optimal Ds concentration for the treatment of cells in vitro, chondrocytes were cultured with Ds gradient for 48 h, followed by cell viability analysis using the CCK-8 assay.The results showed that the cell viability of the group treated with 10% Ds for 48 hours was greater than that of the other four groups (Fig 6C).Chondrocyte viability rate was inhibited by 10 ng/mL of IL-1β, and this effect was reversed by pretreatment with 10% Ds.However, this effect was significantly attenuated after PTEN silencing.Therefore, 10% Ds was used for subsequent experimental studies (Fig 6D).
Next, we examined cell apoptosis in each group using flow cytometry (Fig 7A).The level of apoptosis was reduced significantly after 48-h treatment with Ds, whereas this effect was significantly depressed after PTEN silencing (Fig 7B).Western blotting and RT-PCR analysis of  Beclin-1 were significantly decreased after IL-1β treatment, and the effect was reversed by pretreatment with Ds.Additionally, PTEN silencing weakened Ds-induced increase in the level of autophagy in chondrocytes (Fig 7F -7I).
Reportedly, DHJSD can prevent the degradation of the cartilage matrix by inhibiting MMPs, as revealed in in vivo experiments.Furthermore, zinc is an essential cofactor for a variety of matrix metalloproteinases and is involved in the metabolic homeostasis of cartilage matrix [35,36].Moreover, previous studies have found that DHJSD can decrease zinc levels in chondrocytes and inhibit the expression of MMP-13 and other proteins [19,33].In addition, autophagy has been found to be closely related to intracellular zinc homeostasis [16][17][18].Therefore, we hypothesized that promoting the autophagy of chondrocytes by DHJSD was also related to the intracellular zinc levels.To verify the hypothesis, ICP-MS/MS was used to measure zinc levels in chondrocytes.The results showed that IL-1β treatment increased zinc levels in chondrocytes.However, treatment with Ds significantly reduced intracellular zinc levels, and this effect was significantly reversed after PTEN silencing (Fig 7J).Collectively, these findings indicate that DHJSD decreases zinc levels by upregulating autophagy.

DHJSD regulated the PTEN/Akt/mTOR signaling pathway in vitro
Animal experiments have confirmed that DHJSD can enhance chondrocyte autophagy via PTEN/Akt/mTOR signaling pathway.Hence, we attempted to further elucidate this mechanism in vitro.
Identification of the protein levels of PTEN, p-Akt, Akt, p-mTOR, and mTOR using western blotting revealed that pretreatment with Ds promoted PTEN expression and reduced Akt and mTOR phosphorylation in IL-1β-stimulated chondrocytes.However, silencing PTEN reduced the inhibitory effect of Ds on Akt and mTOR phosphorylation.Notably, Ds demonstrated no significant effect on PTEN expression and Akt and mTOR phosphorylation levels in normal chondrocytes.These findings were additionally confirmed in in vivo experiments (Fig 8).

Discussion
The four major pathological and etiological characteristics of KOA include inflammatory cytokine cascade activation, chondrocyte apoptosis, imbalanced subchondral bone remodeling, and extracellular matrix degradation [37].The majority of the research for studying cartilage degeneration is directed at subchondral bone remodeling, delaying chondrocyte apoptosis, and inhibiting matrix degradation.Currently, non-steroidal anti-inflammatory drugs are the most widely used and effective drugs in the conventional treatment of KOA [38,39].However, the long-term use of these drugs is associated with side effects such as gastrointestinal ulcers and cardiovascular adverse events [40], which are concerning.Moreover, the application of specific treatments for OA, such as glucosamine, hyaluronic acid, and platelet-rich plasma injections, are controversial and either conflict with the guidelines or are not recommended owing to a lack of evidence-based findings regarding the long-term efficacy [41].Therefore, in the current situation, the use of natural compounds, including traditional Chinese medicine formulas, is one of the important directions for the prevention and treatment of KOA.In this regard, DHJSD has a long history of use for the treatment of KOA; however, the mechanism underlying its action has not been fully elucidated.
The soft tissue mechanical abnormality is one of the factors initiating OA [42].Therefore, the classic Hulth method was used to establish the KOA rat model in this study.Reportedly, IL-1β plays a key role in OA pathogenesis [43], and it has been well-established that IL-1β can be used to establish an in vitro cellular OA model.In the present study, both the Hulth method-induced KOA model (in vivo) and IL-1β-induced cellular KOA model (in vitro) substantially replicated human OA-like features.Moreover, treatment of KOA rats with DHJSD attenuated the OA-like features in rats.
The present study investigated the mechanism by which DHJSD prevents and treats KOA in an in vivo rat KOA model and in vitro IL-1β-induced chondrocytes.The results showed that DHJSD could inhibit IL-1β and MMP-13 levels, which was consistent with the results of previous studies [44], thus confirming the anti-inflammatory effect of DHJSD.In addition, the in vivo experiment revealed that DHJSD could alleviate the articular cartilage degradation, repress osteoclast formation, reduce collagen fiber deposition, and prevent type II collagen degradation in the articular cartilage.Moreover, DHJSD could upregulate the protein and mRNA levels of PTEN; inhibit the Akt/mTOR signaling pathway; and promote LC3 and Belcin-1 expression, inhibit Bax and Bcl-2 expression, and reduce zinc levels in chondrocytes.
Autophagy is a key important protective mechanism for cells to avoid self-apoptosis [45,46].Reportedly, the PTEN/Akt signaling axis is closely related to autophagy [20,26,47].In the present study, IL-1β treatment downregulated the expression of PTEN, LC3, and Beclin-1 and upregulated the phosphorylation of Akt and mTOR.In contrast, pretreatment of IL-1βstimulated chondrocytes with Ds increased the expression of PTEN, LC3, and Beclin-1 and decreased the phosphorylation of Akt and mTOR.However, this effect was weakened following the silencing of PTEN.Furthermore, in vivo results demonstrated that the expression of PTEN, LC3, and Beclin-1 in the chondrocytes of KOA rats in the HD group were significantly higher than the expression in the model group, but the levels of p-Akt and p-mTOR were significantly lower than those in the model group.In addition, transmission electron microscopy findings revealed that the formation of autophagosomes in rat chondrocytes in the HD group was significantly higher than that in the model group.Therefore, it is reasonable to speculate that DHJSD can target PTEN to inhibit the Akt/mTOR signaling pathway and upregulate autophagy and LC3 and Beclin-1 protein levels in chondrocytes.
It is well established that MMP-13 plays an important role in the progression of OA [48][49][50], which is the pathological basis for the degradation of articular cartilage.MMPs can be induced via multiple pathways, and inflammatory cytokines such as IL-6 can directly induce the production of MMPs [51].The present study showed that DHJSD directly inhibited the formation of IL-1β and MMP-13 in vivo, thus inducing a protective role for the articular cartilage.Studies have reported that zinc is closely related to the formation of MMPs [52,53].This implies that when cells synthesize more MMPs in an inflammatory state, more zinc is needed to enter the cells.
The present study showed that the intracellular zinc level in chondrocytes increased after IL-1β treatment of chondrocytes, and pretreatment with Ds decreased the zinc level in IL-1βstimulated chondrocytes.These findings indicate that one of the reasons DHJSD may inhibit MMP formation is by downregulating intracellular zinc levels.Thus, DHJSD can enhance the level of autophagy in chondrocytes while also reducing zinc levels in chondrocytes.However, the study could not clarify how zinc is expelled out of cells through autophagy and whether it is related to the cell exocytosis mechanism (Fig 9).The findings of this study indicate that the DHJSD maintains intracellular zinc homeostasis by enhancing cellular autophagy; however, further studies are required to clarify this mechanism.

Conclusion
Histopathological observations verified the improvement in KOA in rats following treatment with DHJSD.Additionally, in the rats with KOA induced using the Hulth method, DHJSD enhanced autophagy by inhibiting the Akt/mTOR signaling pathway by upregulating PTEN protein levels as well as downregulated the levels of proinflammatory cytokines IL-1β and MMP-13.Moreover, in vitro findings revealed that DHJSD can enhance the level of autophagy and regulate intracellular zinc homeostasis through the PTEN/Akt/mTOR signaling pathway.Thus, DHJSD has anti-OA and cartilage protective effects in the treatment of KOA both in vivo and in vitro.

Fig 1 .
Fig 1. Establishment of rat KOA model using the Hulth method.(A) Incision site; (B) exposure of the patellar ligament; (C) cutting of the patellar ligament and exposure of the medial collateral ligament; (D) cutting of the medial collateral ligament and exposure of the medial meniscus; (E) removal of the medial meniscus and exposure of the cruciate ligament; (F) cutting of the cruciate ligament and retention of the lateral meniscus; (G) image showing cut medial collateral ligament and cruciate ligaments, removed medial meniscus, and fully retained lateral meniscus; (H) suturing of incision.https://doi.org/10.1371/journal.pone.0290925.g001 shown in Fig 4A,the model group demonstrated a discontinuous cell membrane, concentrated cytoplasm, and lysed nucleus; moreover, the cytoplasmic content was missing in some areas, and the autophagosomes were hardly observed.Although the HD group demonstrated dilated rough endoplasmic reticulum in some cells, the morphological structure of cells and mitochondria was almost normal, with more autophagosomes in the cytoplasm.Treatment with DHJSD demonstrated a significant, dose-dependent increase in autophagic vesicles in the cytoplasm.At the same time, DHJSD significantly increased the protein levels of LC3 and Beclin-1 and the mRNA expression of Beclin-1 (Fig 4C, 4D and 4G).In contrast, DHJSD decreased the protein level and mRNA expression of Bax and Bcl-2 (Fig 4E-4I).Taken together, DHJSD can increase the level of autophagy and inhibit the apoptosis of articular chondrocytes in KOA rats.